NAD(P)H Oxidase Mediates TGF-β1–Induced Activation of Kidney Myofibroblasts

TGF-β1 expression closely associates with activation and conversion of fibroblasts to a myofibroblast phenotype and synthesis of an alternatively spliced cellular fibronectin variant, Fn-ED-A. Reactive oxygen species (ROS), such as superoxide, which is a product of NAD(P)H oxidase, also promote the transition of fibroblasts to myofibroblasts, but whether these two pathways are interrelated is unknown. Here, we examined a role for NAD(P)H oxidase–derived ROS in TGF-β1–induced activation of rat kidney fibroblasts and expression of α-smooth muscle actin (α-SMA) and Fn-ED-A. In vitro, TGF-β1 stimulated formation of abundant stress fibers and increased expression of both α-SMA and Fn-ED-A. In addition, TGF-β1 increased both the activity of NADPH oxidase and expression of Nox2 and Nox4, homologs of the NAD(P)H oxidase family, indicating that this growth factor induces production of ROS. Small interfering RNA targeted against Nox4 markedly inhibited TGF-β1–induced stimulation of NADPH oxidase activity and reduced α-SMA and Fn-ED-A expression. Inhibition of TGF-β1 receptor 1 blocked Smad3 phosphorylation; reduced TGF-β1–enhanced NADPH oxidase activity; and decreased expression of Nox4, α-SMA, and Fn-ED-A. Diphenyleneiodonium, an inhibitor of flavin-containing enzymes such as the Nox oxidases, had no effect on TGF-β1–induced Smad3 but reduced both α-SMA and Fn-ED-A protein expression. The Smad3 inhibitor SIS3 reduced NADPH oxidase activity, Nox4 expression, and blocked α-SMA and Fn-ED-A, indicating that stimulation of myofibroblast activation by ROS is downstream of Smad3. In addition, TGF-β1 stimulated phosphorylation of extracellular signal–regulated kinase (ERK1/2), and this was inhibited by blocking TGF-β1 receptor 1, Smad3, or the Nox oxidases; ERK1/2 activation increased α-SMA and Fn-ED-A. Taken together, these results suggest that TGF-β1–induced conversion of fibroblasts to a myofibroblast phenotype involves a signaling cascade through Smad3, NAD(P)H oxidase, and ERK1/2.Progression of renal fibrosis involves expansion of interstitial myofibroblasts and extracellular matrix accumulation, resulting in the loss of function and ultimately renal failure.^1,2 The origin of myofibroblasts is under extensive investigation, and evidence indicates the cells may be derived from several sources, including an expansion of activated resident fibroblasts, perivascular adventitial cells, blood-borne stem cells that migrate into the glomerular mesangial or interstitial compartment, or tubular epithelial-to-mesenchymal transition and migration into the peritubular interstitial space. Regardless of their origin, there is common agreement that the myofibroblast is the cell most responsible for interstitial expansion and matrix accumulation during the course of renal fibrosis. TGF-β1 is the predominant growth factor responsible for matrix synthesis by mesenchymal cells such as fibroblasts in vitro and during renal fibrosis.^3,4 Indeed, there is a close correlation in the cellular expression of TGF-β1, a fibroblast transition to an activated, α-smooth muscle actin (α-SMA)-positive myofibroblast phenotype, and synthesis of an alternatively spliced isoform of fibronectin, Fn-ED-A.⁵ TGF-β1 differentially regulates the expression of Fn-ED-A in fibroblasts^6–8 and induces expression of α-SMA in a variety of mesenchymal cells in culture.^9,10 Indeed, a functional ED-A domain is mandatory for α-SMA induction by TGF-β1.^7,8,10 Moreover, TGF-β1 is frequently associated with a myofibroblast phenotype in liver, lung, and kidney disease,^1,11–13 and all three proteins frequently co-localize in these disease settings. In addition, a co-localization of α-SMA and Fn-ED-A is frequently observed in fibrotic disease as well as in glomerular and interstitial lesions in kidney diseases previously investigated in our laboratory.^14–17Accumulating evidence also indicates that reactive oxygen species (ROS), mainly in the form of superoxide, play a significant role in the initiation and progression of cardiovascular^18,19 and renal^20–25 disease. ROS are involved in distinct cell functions, including hypertrophy, migration, proliferation, apoptosis, and regulation of extracellular matrix.^25–28 More specific, the NAD(P)H oxidases of the Nox family have gained heightened attention as mediators of injury associated with vascular diseases, including hypertension, atherosclerosis, heart disease, and diabetes.^18,19,29,30 NAD(P)H oxidase generation of superoxide is recognized as an important mediator of cell proliferation in glomerulonephritis²² and matrix accumulation in diabetic nephropathy^25,31–33 and fibrosis.^21,24 Adventitial fibroblasts are also a major source of superoxide in the aorta,^19,34–36 therefore being highly relevant to renal disease. This is because the renal perivascular space is noticeably reactive and is the site where myofibroblasts may first appear during the course of renal disease and fibrosis.^17,37–39The observations that both TGF-β1 and ROS induce fibroblasts to α-SMA–positive myofibroblast phenotype^40–42 suggest that these two pathways are interrelated and may share signaling pathways in kidney disease. TGF-β signaling occurs through a well-established process involving two downstream pathways: Smad and extracellular signal–regulated kinase (ERK).^43–45 TGF-β/Smad signaling (Smad2 and Smad3) is tightly controlled by mitogen-activated protein kinase (MAPK; ras/MEK/ERK) signaling cascades.⁴⁶ A regulatory role for ROS in PDGF and angiotensin II–induced signal transduction has gained recognition^47,48; however, a role for ROS in TGF-β signaling is less well understood. It is also unknown whether kidney myofibroblasts express NAD(P)H oxidase homologs or generate ROS in response to TGF-β1. Given TGF-β1–induced myofibroblast activation and matrix synthesis during renal disease may be linked to ROS, we examined a role for NAD(P)H oxidase in TGF-β1–induced Smad3 and ERK signaling as well as kidney myofibroblast activation, as assessed by a switch to an α-SMA–positive phenotype and expression of Fn-ED-A expression in vitro.     

Direct Effects of TNF-α on Local Fuel Metabolism and Cytokine Levels in the Placebo-Controlled,Bilaterally Infused Human Leg: Increased Insulin Sensitivity,Increased Net Protein Breakdown,and Increased IL-6 Release

Tumor necrosis factor-α (TNF-α) has widespread metabolic actions. Systemic TNF-α administration, however, generates a complex hormonal and metabolic response. Our study was designed to test whether regional, placebo-controlled TNF-α infusion directly affects insulin resistance and protein breakdown. We studied eight healthy volunteers once with bilateral femoral vein and artery catheters during a 3-h basal period and a 3-h hyperinsulinemic-euglycemic clamp. One artery was perfused with saline and one with TNF-α. During the clamp, TNF-α perfusion increased glucose arteriovenous differences (0.91 ± 0.17 vs. 0.74 ± 0.15 mmol/L, P = 0.012) and leg glucose uptake rates. Net phenylalanine release was increased by TNF-α perfusion with concomitant increases in appearance and disappearance rates. Free fatty acid kinetics was not affected by TNF-α, whereas interleukin-6 (IL-6) release increased. Insulin and protein signaling in muscle biopsies was not affected by TNF-α. TNF-α directly increased net muscle protein loss, which may contribute to cachexia and general protein loss during severe illness. The finding of increased insulin sensitivity, which could relate to IL-6, is of major clinical interest and may concurrently act to provide adequate tissue fuel supply and contribute to the occurrence of systemic hypoglycemia. This distinct metabolic feature places TNF-α among the rare insulin mimetics of human origin.Originally, tumor necrosis factor-α (TNF-α) was identified as an endogenous pyrogen or “cachectin” (1) because of its biological properties of inducing fever, cachexia, and muscle protein loss in various states of disease (2–4). TNF-α is a key component of an inflammatory response and one of the most potent proinflammatory cytokines released by innate immune cells that induces release of other cytokines, including interleukin-6 (IL-6) (5,6). TNF-α plays an important role in the pathophysiology of sepsis, and there seems to be a relation between the TNF-α level and the severity of disease (7–9). Finally, TNF-α has been associated with states of constant low-grade inflammation, eventually leading to insulin resistance and overt diabetes (10,11). In line with this, it has been shown that plasma levels of TNF-α are correlated with BMI; weight loss leads to a decrease in plasma levels of TNF-α (12,13).Systemic infusion of TNF-α induces insulin resistance and increased lipolysis in humans (6,14,15), whereas the effects on protein metabolism are less clear (16). A number of studies have shown that anti–TNF-α treatment increases insulin sensitivity in patients with inflammatory chronic diseases (17–19), whereas other reports have failed to confirm this relationship (20–23). Furthermore, studies investigating TNF-α neutralization in type 2 diabetic patients and in patients with metabolic syndrome show no effect of anti–TNF-α treatment on insulin sensitivity (24,25). TNF-α activates the hypothalamopituitary axis and stimulates the release of stress hormones, such as epinephrine, glucagon, cortisol, and growth hormone into the blood (26,27); all of these counter-regulatory stress hormones generate insulin resistance (27–29), and glucocorticoids generate muscle loss (30). Thus, TNF-α invariably generates release of both other cytokines and stress hormones, and it is uncertain to which extent the metabolic actions of TNF-α are intrinsic or caused by other cytokines or stress hormones in humans.The current study was therefore designed to define the direct metabolic effects of TNF-α in human muscle. Since all previous human studies assessing the metabolic actions of TNF-α have used systemic administration, making discrimination between direct and indirect effects impossible, we infused TNF-α directly into the femoral artery and compared the effects to the saline-infused contralateral leg.     

Pathogenesis of A?β+ Ketosis-Prone Diabetes

A⁻β⁺ ketosis-prone diabetes (KPD) is an emerging syndrome of obesity, unprovoked ketoacidosis, reversible β-cell dysfunction, and near-normoglycemic remission. We combined metabolomics with targeted kinetic measurements to investigate its pathophysiology. Fasting plasma fatty acids, acylcarnitines, and amino acids were quantified in 20 KPD patients compared with 19 nondiabetic control subjects. Unique signatures in KPD—higher glutamate but lower glutamine and citrulline concentrations, increased β-hydroxybutyryl-carnitine, decreased isovaleryl-carnitine (a leucine catabolite), and decreased tricarboxylic acid (TCA) cycle intermediates—generated hypotheses that were tested through stable isotope/mass spectrometry protocols in nine new-onset, stable KPD patients compared with seven nondiabetic control subjects. Free fatty acid flux and acetyl CoA flux and oxidation were similar, but KPD had slower acetyl CoA conversion to β-hydroxybutyrate; higher fasting β-hydroxybutyrate concentration; slower β-hydroxybutyrate oxidation; faster leucine oxidative decarboxylation; accelerated glutamine conversion to glutamate without increase in glutamate carbon oxidation; and slower citrulline flux, with diminished glutamine amide–nitrogen transfer to citrulline. The confluence of metabolomic and kinetic data indicate a distinctive pathogenic sequence: impaired ketone oxidation and fatty acid utilization for energy, leading to accelerated leucine catabolism and transamination of α-ketoglutarate to glutamate, with impaired TCA anaplerosis of glutamate carbon. They highlight a novel process of defective energy production and ketosis in A⁻β⁺ KPD.Ketosis-prone diabetes (KPD) is characterized by presentation with diabetic ketoacidosis (DKA) in persons who do not fit traditional categories of types 1 or 2 diabetes (1–5). We have defined four subgroups of KPD based on presence or absence of β-cell autoantibodies (A⁺ or A⁻), and recovery or lack of recovery of β-cell functional reserve following the index episode of DKA (β⁺ or β⁻) (1,6,7).The A⁻β⁺ KPD subgroup represents a novel syndrome of severe but reversible β-cell dysfunction (1,3,5,8,9). Approximately 50% of these patients develop DKA without a precipitating factor at diagnosis of diabetes. These new-onset, unprovoked A⁻β⁺ KPD patients display male predominance (10) and low frequencies of human leukocyte antigen class II susceptibility alleles for autoimmune diabetes (11). β-Cell function increases markedly within 1–3 months after the index DKA, with sustained glycemic improvement and insulin independence (1,9,11,12). The cause of the unprovoked ketoacidosis is unknown. Over 5–10 years, patients may relapse to unprovoked ketosis (3,5).This syndrome provides a model to identify novel mechanisms of obesity, ketosis, and reversible β-cell dysfunction. We used a metabolomics approach to identify unique alterations in A⁻β⁺ KPD patients, with a kinetics approach to specify the pathophysiology.     

C-Peptide Activates AMPKα and Prevents ROS-Mediated Mitochondrial Fission and Endothelial Apoptosis in Diabetes

Vasculopathy is a major complication of diabetes; however, molecular mechanisms mediating the development of vasculopathy and potential strategies for prevention have not been identified. We have previously reported that C-peptide prevents diabetic vasculopathy by inhibiting reactive oxygen species (ROS)-mediated endothelial apoptosis. To gain further insight into ROS-dependent mechanism of diabetic vasculopathy and its prevention, we studied high glucose–induced cytosolic and mitochondrial ROS production and its effect on altered mitochondrial dynamics and apoptosis. For the therapeutic strategy, we investigated the vasoprotective mechanism of C-peptide against hyperglycemia-induced endothelial damage through the AMP-activated protein kinase α (AMPKα) pathway using human umbilical vein endothelial cells and aorta of diabetic mice. High glucose (33 mmol/L) increased intracellular ROS through a mechanism involving interregulation between cytosolic and mitochondrial ROS generation. C-peptide (1 nmol/L) activation of AMPKα inhibited high glucose–induced ROS generation, mitochondrial fission, mitochondrial membrane potential collapse, and endothelial cell apoptosis. Additionally, the AMPK activator 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside and the antihyperglycemic drug metformin mimicked protective effects of C-peptide. C-peptide replacement therapy normalized hyperglycemia-induced AMPKα dephosphorylation, ROS generation, and mitochondrial disorganization in aorta of diabetic mice. These findings highlight a novel mechanism by which C-peptide activates AMPKα and protects against hyperglycemia-induced vasculopathy.C-peptide and insulin are cosecreted in equimolar amounts into the circulation from the pancreatic β-cells of Langerhans (1). C-peptide deficiency is a prominent attribute of type 1 diabetes (1). Deficiencies of C-peptide and insulin may also occur in the late stages of type 2 diabetes as a result of progressive loss of β-cells (2–4). Recent evidence demonstrates a beneficial role for C-peptide in diabetic neuropathy (1,5,6), nephropathy (1,6,7), and vascular dysfunction (1,5) and inflammation (1). C-peptide protects against diabetic vascular damage by promoting nitric oxide (NO) release (8) and suppressing nuclear factor-κB (9), which suppresses leukocyte-endothelium interactions (8,9). C-peptide may prevent atherosclerosis by inhibiting vascular smooth muscle proliferation and migration (10) and reducing venous neointima formation (11). However, the molecular mechanism by which C-peptide prevents diabetes complications is not understood well enough to permit its clinical implementation.Generation of reactive oxygen species (ROS) in response to high glucose is the leading cause of endothelial damage and diabetic vasculopathy (12). Protein kinase C (PKC)-dependent NADPH oxidase is considered a major cytosolic mediator of ROS generation in endothelial cells (13,14) that play a central role in hyperglycemia-induced endothelial cell apoptosis and vascular complications (15–17). Overproduction of intracellular ROS by mitochondria also occurs during the development of hyperglycemia-induced vascular complications (12,18,19). Altered mitochondrial dynamics due to mitochondrial fission were recently linked with endothelial dysfunction in diabetes (20,21). However, the mechanisms regulating production of cytosolic and mitochondrial ROS and their individual functions in regulating mitochondrial dynamics and apoptosis remain to be elucidated.AMP-activated protein kinase (AMPK) is an intracellular energy and stress sensor (22) and is an emerging target for preventing diabetes complications (23), as exhibited by the most common antihyperglycemic drugs, rosiglitazone (24) and metformin (25). AMPK prevents apoptosis of endothelial cells (26–28) by inhibiting ROS generation by NADPH oxidase (24,29) and mitochondria (30). Additionally, AMPK dephosphorylation is associated with diabetes (22,31,32). It has been reported that C-peptide inhibits high glucose–induced mitochondrial superoxide generation in renal microvascular endothelial cells (7). We recently demonstrated a key role for C-peptide in preventing high glucose–induced ROS generation and apoptosis of endothelial cells through inhibition of transglutaminase (17). However, the mechanism underlying C-peptide–mediated inhibition of intracellular ROS production and subsequent apoptosis remains unclear. Thus, we hypothesized that the potential protective role of C-peptide could be attributed to activation of AMPK, which results in reduced hyperglycemia-induced production of intracellular ROS and altered mitochondrial dynamics that suppress apoptosis of endothelial cells.In this study, we sought to elucidate the mechanism by which C-peptide protects against hyperglycemia-induced ROS production and subsequent endothelial cell damage. We examined the beneficial effect of C-peptide through AMPKα activation and subsequent protection against hyperglycemia-induced production of intracellular ROS, dysregulation of mitochondrial dynamics, mitochondrial membrane potential (∆Ψ_m) collapse, and apoptosis of endothelial cells. These studies were confirmed in vivo in mice with streptozotocin-induced diabetes using C-peptide supplement therapy delivered through osmotic pumps. Thus, our study implicates C-peptide replacement therapy as a potentially significant approach for preventing diabetes complications.     

Cyclic GMP kinase I modulates glucagon release from pancreatic α-cells

OBJECTIVE

The physiologic significance of the nitric oxide (NO)/cGMP signaling pathway in islets is unclear. We hypothesized that cGMP-dependent protein kinase type I (cGKI) is directly involved in the secretion of islet hormones and glucose homeostasis.

RESEARCH DESIGN AND METHODS

Gene-targeted mice that lack cGKI in islets (conventional cGKI mutants and cGKIα and Iβ rescue mice [α/βRM] that express cGKI only in smooth muscle) were studied in comparison to control (CTR) mice. cGKI expression was mapped in the endocrine pancreas by Western blot, immuno-histochemistry, and islet-specific recombination analysis. Insulin, glucagon secretion, and cytosolic Ca²⁺ ([Ca²⁺]_i) were assayed by radioimmunoassay and FURA-2 measurements, respectively. Serum levels of islet hormones were analyzed at fasting and upon glucose challenge (2 g/kg) in vivo.

RESULTS

Immunohistochemistry showed that cGKI is present in α- but not in β-cells in islets of Langerhans. Mice that lack α-cell cGKI had significantly elevated fasting glucose and glucagon levels, whereas serum insulin levels were unchanged. High glucose concentrations strongly suppressed the glucagon release in CTR mice, but had only a moderate effect on islets that lacked cGKI. 8-Br-cGMP reduced stimulated [Ca²⁺]_i levels and glucagon release rates of CTR islets at 0.5 mmol/l glucose, but was without effect on [Ca²⁺]_i or hormone release in cGKI-deficient islets.

CONCLUSIONS

We propose that cGKI modulates glucagon release by suppression of [Ca²⁺]_i in α-cells.The complex and tightly controlled process of glucagon secretion from pancreatic α-cells is important for the maintenance of blood glucose homeostasis (1). Glucagon release is physiologically regulated by multiple signaling pathways that include neuronal control of α-cell function, paracrine factors such as insulin (2,3), and/or γ-aminobutyric acid (GABA) (4) released from β-cells, somatostatin (SST) secreted from adjacent δ-cells (5), and the inhibitory role of high blood glucose itself that directly acts on α-cells to suppresses glucagon release (3,6).Controversial data have been reported for the physiologic significance of the nitric oxide (NO) pathway for islet functions. Both types of constitutive NOS (eNOS, nNOS) isozymes have been identified in islets (7–11). It was suggested that NO stimulates glucose-induced insulin release (7,10), was a negative modulator of insulin release (8,12,13) or had no effect (14). These discrepant results are probably caused by the analysis of various β-cell–derived cell lines compared with intact islets, the use of different types and concentrations of NO-donors and NOS-inhibitors. Additional data suggested that iNOS-derived NO is involved in autoimmune reactions that cause β-cell–dysfunction leading to insulin-dependent diabetes (15,16).It has been difficult to discriminate between a direct action of NO on α-cells and an indirect effect of NO via β-cells since β-cell factors are potent inhibitors of α-cell activity (12,17,18). An important target of NO is the soluble guanylyl cyclase (sGC) that generates the second messenger cyclic guanosine-3′-5′-monophosphate (cGMP) (19). Some studies detected increased islet cGMP levels upon treatment with cytokines and l-arginine (12,15,20). cGMP analogues were reported to potentiate insulin release directly (21), suggesting that cGMP-dependent effectors are involved in the control of islet activity. The cGMP-dependent protein kinase type I (cGKI) is an important intracellular mediator of NO/cGMP signaling in many cells (22). The analysis of cGKI-deficient mice revealed that cGKI mediates the inhibitory effects of NO on platelet aggregation, the negative inotropic effect of NO/cGMP in the murine heart, and the NO-induced relaxation of blood vessels (22). However, cGKI knockout mice could not be analyzed reliably for a distorted islet function because they display abnormalities of various organ systems and die within the first 6 weeks (23). Recently, we generated an improved mouse model to study the specific roles of cGKI in vivo (24,25). These mice express either the cGKIα or cGKIβ isozyme selectively in smooth muscle cells (SMCs) on a cGKI-deficient genetic background. Since the animals show a prolonged life expectancy and normal SMC functions they were termed cGKIα and cGKIβ rescue mice (αRM and βRM, respectively).We examined the role of cGKI for the regulation of glucose homeostasis using cGKI-KO mice (23) and rescue mice (RM) (24,25) models. We show that cGKI is expressed in the α-cells of the endocrine pancreas, whereas in different gene-targeted animals the cGKI protein was not detectable. Furthermore, we demonstrate that islet cGKI regulates glucagon release by modulation of the glucose-dependent changes of [Ca²⁺]_i that trigger exocytosis. These ex-vivo findings were supported by elevated serum levels of basal glucose and glucagon in intact RM animals.     

Advanced Glycation End-Products Induce Tubular CTGF via TGF-β–Independent Smad3 Signaling

Advanced glycation end-products (AGEs) can induce expression of connective tissue growth factor (CTGF), which seems to promote the development of diabetic nephropathy, but the exact signaling mechanisms that mediate this induction are unknown. Here, AGEs induced CTGF expression in tubular epithelial cells (TECs) that either lacked the TGF-β1 gene or expressed dominant TGF-β receptor II, demonstrating independence of TGF-β. Furthermore, conditional knockout of the gene encoding TGF-β receptor II from the kidney did not prevent AGE-induced renal expression of CTGF and collagen I. More specific, AGEs induced CTGF expression via the receptor for AGEs-extracellular signal–regulated kinase (RAGE-ERK)/p38 mitogen-activated protein kinase–Smad cross-talk pathway because inhibition of this pathway by several methods (anti-RAGE antibody, specific inhibitors, or dominant negative adenovirus to ERK1/2 and p38) blocked this induction. Overexpressing Smad7 abolished AGE-induced Smad3 phosphorylation and CTGF expression, demonstrating the necessity for activation of Smad signaling in this process. More important, knockdown of either Smad3 or Smad2 demonstrated that Smad3 but not Smad2 is essential for CTGF induction in response to AGEs. In conclusion, AGEs induce tubular CTGF expression via the TGF-β–independent RAGE-ERK/p38-Smad3 cross-talk pathway. These data suggest that overexpression of Smad7 or targeting Smad3 may have therapeutic potential for diabetic nephropathy.Connective tissue growth factor (CTGF; CCN2), a member of CCN family of growth factors, plays an important role in connective tissue homeostasis and fibroblast proliferation, migration, adhesion, and extracellular matrix expression.¹ Clinically, renal expression of CTGF is increased in patients with diabetic nephropathy (DN), and its expression correlates closely with the degree of albuminuria.^2,3 In addition, studies in human renal biopsy show that CTGF expression significantly augments glomerular and tubulointerstitial injury with α-smooth muscle actin cell accumulation.⁴ Several pieces of evidence from recent rodent studies further support the notion that CTGF is important in the pathogenesis of DN. For example, the thickening of glomerular basement membrane is attenuated in CTGF^+/− mice.⁵ In type 1 diabetic mouse model, cell-specific overexpression of CTGF in podocytes of CTGF transgenic mice is able to intensify proteinuria and mesangial expansion.⁶ The co-localization of increased renal CTGF expression and AGE accumulation in diabetic rats indicates a causal link between AGE deposition and CTGF expression.⁷ This is supported by the ability of the AGE inhibitor to suppress CTGF expression and reduce renal fibrosis.⁷ Although the mechanisms that regulate renal CTGF function are not clearly understood, CTGF should play an essential role in DN.Engagement of AGEs to the receptor (RAGE) has been shown to play a critical role in diabetic complications, including DN.⁸ Indeed, AGE-induced tubular epithelial-to-mesenchymal transition (EMT) and renal fibrosis are RAGE dependent.^8,9 Under diabetic conditions, although treatments with high glucose and angiotensin II are also able to upregulate CTGF expression in glomerular mesangial cells (MCs) and TECs,^2,10–12 it is clear that AGEs mediate CTGF expression by stimulating TGF-β expression.^13,14 It is generally believed that TGF-β/Smad signaling should be responsible for inducing CTGF expression because CTGF is a downstream mediator of TGF-β signaling^{11,12,15–17}; however, the exact mode of signaling mechanisms by which AGEs induce CTGF expression remains largely unclear.Our previous study of MCs, TECs, and vascular smooth muscle cells (VSMCs) showed that AGEs are able to induce Smad2/3 phosphorylation markedly in TGF-β receptor I (TβRI) and TβRII mutant cell lines via the extracellular signal–regulated kinase (ERK)/p38 mitogen-activate protein kinase (MAPK)-dependent mechanism.¹⁸ This demonstrates a critical role for the TGF-β–independent Smad pathway in AGE-mediated fibrotic response. This is further supported by the finding that blockade of TGF-β1 with specific small hairpin RNA (shRNA) and a neutralizing antibody is unable to inhibit significantly AGE-induced CTGF mRNA expression.¹⁹ All of these studies suggest a TGF-β–independent mechanism in regulating CTGF expression in response to AGEs. Because AGEs are capable of activating the TGF-β/Smad signaling pathway via the ERK/p38 MAPK-dependent mechanism and because CTGF is a target gene of TGF-β/Smad signaling,^7,15,19–22 we thus hypothesized that AGEs might induce CTGF expression via the TGF-β–independent Smad3 signaling pathway. This was tested in mouse TECs lacking TGF-β1 gene²³ and rat TEC lines overexpressing the dominant negative TβRII or Smad7 or having a knockdown of Smad2 or Smad3. Finally, the functional importance of the TGF-β–independent signaling pathway in AGE-mediated CTGF expression and renal fibrosis was tested in mice that had conditional knockout (KO) for TβRII from the kidney.